Normal human somatic cells have a limited capacity to proliferate, a process termed replicative senescence. Increasing evidence over the last decade has implicated telomeres, the structures that cap the ends of the chromosomes, as the molecular clock that counts the number of times the cell has divided. The mechanism of lagging strand DNA synthesis prevents DNA polymerase from replicating the DNA all the way to the 3'end of a linear chromosome, causing the chromosomes to shorten every time a cell divides. Telomeres prevent the cell from recognizing the end of the chromosome as a DNA break needing repair. Cellular senescence occurs when some of the telomeres have shortened sufficiently to induce a DNA damage signal. Cellular immortalization is usually accompanied by the reactivation of the enzyme telomerase, which is able to add telomeric repeats to the ends of the chromosomes and thus prevent their shortening. In rare cases, a recombination-based alternative lengthening of telomeres (ALT) pathway is used to maintain telomeres. This proposal consists of three broad aims: Aim 1 is directed towards understanding the molecular basis for telomere position effects, including the identification of sequences that influence the propagation or inhibition of silencing, and the endogenous genes whose expression is regulated by telomere length;Aim 2 exploits our identification of a molecular assay for the ALT phenotype to screen for factors influencing recombination within telomeric DNA. Finally, Aim 3 proposes experiments to define the molecules regulating the transcription and action/recruitment of telomerase on telomeres. The information resulting from these studies should help identify the molecular mechanisms underlying replicative senescence and its relationship to cancer. Lay Summary: Normal cells do not divide forever, in contrast to cancer cells. Cancer cells have become immortal because they have activated mechanisms that maintain the ends of their chromosomes, structures called telomeres. This proposal investigates the mechanisms by which cancer cells maintain their telomeres, and normal cells use their telomeres to regulate their behavior. Insights from these studies may lead to treatments for cancer and interventions to modify cellular aging.